U.S. patent application number 13/756053 was filed with the patent office on 2013-11-28 for method for detecting escherichia coli.
This patent application is currently assigned to SYSTAGENIX WOUND MANAGEMENT (US), INC.. The applicant listed for this patent is Systagenix Wound Management (US), Inc.. Invention is credited to Gerard J. Colpas, Diane L. Ellis-Busby, Mitchell C. Sanders, Shite Sebastian.
Application Number | 20130316369 13/756053 |
Document ID | / |
Family ID | 33131532 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130316369 |
Kind Code |
A1 |
Colpas; Gerard J. ; et
al. |
November 28, 2013 |
METHOD FOR DETECTING ESCHERICHIA COLI
Abstract
Described herein are methods of detecting an infection and for
detecting the presence or absence of microorganisms, for example,
wound pathogens in a sample, by contacting a sample with an enzyme
produced and/or secreted by the bacteria, and detecting
modification or the absence of modification or the substrate, as an
indicator of the presence or absence of the enzyme in the sample.
The present invention also features a biosensor for detecting the
presence or absence of bacteria in a sample.
Inventors: |
Colpas; Gerard J.; (Holden,
MA) ; Ellis-Busby; Diane L.; (Lancaster, MA) ;
Sebastian; Shite; (Worcester, MA) ; Sanders; Mitchell
C.; (West Boylston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Systagenix Wound Management (US), Inc.; |
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US |
|
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Assignee: |
SYSTAGENIX WOUND MANAGEMENT (US),
INC.
Wilmington
DE
|
Family ID: |
33131532 |
Appl. No.: |
13/756053 |
Filed: |
January 31, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12894212 |
Sep 30, 2010 |
8377651 |
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13756053 |
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10543554 |
Apr 28, 2006 |
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PCT/US2004/002594 |
Jan 30, 2004 |
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12894212 |
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60444523 |
Jan 31, 2003 |
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Current U.S.
Class: |
435/7.4 |
Current CPC
Class: |
G01N 33/573 20130101;
G01N 33/52 20130101; G01N 2333/245 20130101; C12Q 1/10 20130101;
C12Q 1/37 20130101; G01N 33/56916 20130101; G01N 33/54366
20130101 |
Class at
Publication: |
435/7.4 |
International
Class: |
G01N 33/573 20060101
G01N033/573 |
Claims
1. A method for detecting the presence or absence of an infection
in a subject, comprising the steps of: a) contacting a sample
obtained from a subject with a detectably labeled substrate for an
enzyme produced and/or secreted by Escherichia coli, under
conditions that result in modification of said substrate by said
enzyme; and b) detecting the modification or the absence of the
modification of said substrate, wherein modification of said
substrate indicates the presence of an infection in said subject,
and wherein the absence of modification of said substrate indicates
the absence of an infection in said subject.
2. The method of claim 1, wherein said enzyme is a protease.
3. The method of claim 1, wherein said sample is a body fluid.
4. The method of claim 1, wherein said substrate is on a solid
support.
5. The method of claim 4, wherein said solid support comprises a
material required to be free of microbial contaminants.
6. The method of claim 5, wherein said solid support is selected
from the group consisting of a wound dressing, a container for
holding body fluids, a disk, a scope, a filter, a lens, foam, cloth
paper, a suture, and a swab.
7. The method of claim 6, wherein said container for holding body
fluids is selected from the group consisting of a urine collection
bag, a blood collection bag, a plasma collection bag, a test tube,
a catheter, and a well of a microplate.
8. A method for detecting the presence or absence of a wound
infection in a subject, comprising the steps of: a) contacting a
wound in a subject with a detectably labeled substrate for an
enzyme produced and/or secreted by Escherichia coli under
conditions that result in cleavage of said substrate by said
enzyme, wherein the detectably labeled substrate provides a latent
signal that is activated only when the substrate is cleaved; and b)
measuring the signal to detect the cleavage or the absence of
cleavage of said substrate, wherein cleavage of said substrate
indicates the presence of a wound infection in said subject, and
wherein the absence of cleavage of said substrate indicates the
absence of a wound infection in said subject.
9. The method of claim 8, wherein said enzyme is a protease.
10. The method of claim 8, wherein said substrate is on a solid
support.
11. The method of claim 10, wherein said solid support is a wound
dressing.
12. The method of claim 8, wherein the detectably labeled substrate
is a peptide having a dabcyl moiety and an edans moiety attached at
opposite ends of the peptide.
13. The method of claim 12, wherein the substrate is cleaved
between the dabcyl moiety and the edans moiety.
14. The method of claim 8, wherein the detectably labeled substrate
specific for Escherichia coli is a peptide selected from the group
consisting of: (dabcyl-K)VSRRRRRGG(D-edans) (SEQ ID NO:2),
KKAS(E-edans)VSRRRRRGG(K-dabcyl) (SEQ ID NOS: 3 and 4), and
CHHHAS(E-edans)VSRRRRRGG(K-dabcyl) (SEQ ID NOS: 5 and 6).
15. The method of claim 14, wherein the peptide has an affinity tag
for the non-covalent or covalent attachment of the peptide
substrate to a surface.
16. The method of claim 10, wherein said solid support comprises a
material required to be free of microbial contaminants.
17. A method for detecting the presence or absence of a wound
infection in a subject, comprising the steps of: a) contacting a
wound in a subject with a detectably labeled substrate for an
enzyme produced and/or secreted by Escherichia coli, under
conditions that result in modification of said substrate by said
enzyme; and b) detecting the presence of a known color resulting
from the modification or the absence of the modification of said
substrate, wherein modification of said substrate indicates the
presence of a wound infection in said subject, and wherein the
absence of modification of said substrate indicates the absence of
a wound infection in said subject.
18. The method of claim 17, wherein the known color is produced by
the hydrolysis of the detectably labeled substrate.
19. The method of claim 18, wherein the detectable label on the
substrate is a chromogenic dye.
20. The method of claim 19, wherein the chromogenic dye is
para-nitrophenol.
Description
RELATED APPLICATIONS
[0001] This present application is a continuation of U.S.
application Ser. No. 12/894,212, filed on Sep. 30, 2010, which is a
divisional of U.S. application Ser. No. 10/543,554, filed Apr. 28,
2006, which is a U.S. National Stage Entry of PCT/US2004/002594,
filed Jan. 30, 2004, which claims priority to U.S. Provisional
Application Ser. No. 60/444,523, filed Jan. 31, 2003, all of which
are hereby incorporated by reference in their entireties.
REFERENCE TO A "SEQUENCE LISTING"
[0002] The sequence listing submitted via EFS, in compliance with
37 CFR .sctn.1.52(e)(5), is incorporated herein by reference. The
sequence listing text file submitted via EFS contains the file
"SeqListing.sub.--000119_ST125", created on Jan. 30, 2013, which is
1,653 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Infections are a major source of health expenditure.
Approximately 5% of all surgical wounds become infected with
microorganisms, and that figure is considerably higher (10-20%) for
patients undergoing abdominal surgery. Bacterial species, such as
Escherichia coli (E. coli) are the most frequently isolated
organisms from infected wounds. Bacterial colonization rates are
significantly higher in the hospital setting, both among healthcare
workers, and among patients. Moreover, the colonizing organisms in
the hospital environment are likely to be resistant to many forms
of antimicrobial therapy, due to the strong selective pressure that
exists in the nosocomial environment, where antibiotics are
frequently used. Most strains of Escherichia coli can harmlessly
coexist with humans, for example, in their intestines, and are not
likely to cause disease under normal circumstances. Some strains,
however, produce toxins that can cause severe, even life
threatening disorders, including intestinal disorders, kidney
disorders, and urinary tract infections.
[0004] Escherichia coli are one type of pathogenic microorganism
that can be found in infections in the human body; others include,
but are not limited to Streptococcus pyogenes, Pseudomonas
aeruginosa, Enterococcus faecalis, Proteus mirabilis, Serratia
marcescens, Enterobacter clocae, Acetinobacter anitratus,
Klebsiella pneumoniae, and Staphylococcus species.
[0005] Infection, including wound infection due to any of the above
organisms is a significant concern of hospitals. The most common
way of preventing such infection is to administer prophylactic
antibiotic drugs. While generally effective, this strategy has the
unintended effect of breeding resistant strains of bacteria. The
routine use of prophylactic antibiotics should be discouraged for
the very reason that it encourages the growth of resistant
strains.
[0006] Rather than using routine prophylaxis, a better approach is
to practice good anti-microbial management, i.e., keep area at risk
for becoming infected away from bacteria before, during, and after
surgery, and carefully monitor the wound site or patient fluid for
infection. Normal monitoring methods include close observation of
the wound site for slow healing, signs of inflammation and pus, as
well as measuring the patient's temperature for signs of fever and
testing the patient's fluids, for example, urine, for signs of
infection. Unfortunately, many symptoms are only evident after the
infection is already established. Furthermore, after a patient is
discharged from the hospital they become responsible for monitoring
their own healthcare, and the symptoms of infection may not be
evident to the unskilled patient.
[0007] A system or biosensor that can detect the early stages of
infection before symptoms develop would be advantageous to both
patients and healthcare workers. If a patient can accurately
monitor the condition of a wound after discharge, then appropriate
antimicrobial therapy can be initiated early enough to prevent a
more serious infection.
BRIEF SUMMARY OF THE INVENTION
[0008] It has been found that molecules, for example, proteins
secreted by microorganisms, such as bacteria or fungi, expressed on
the cell surface of microorganisms, or expressed on the surface of
a cell infected with a virus or prion can serve as markers for the
detection of the presence or absence of the microorganism in a
sample, for example, a wound or body fluid. Accordingly, the
present invention features a method of detecting the presence or
absence of a microorganism, for example, E. coli in a sample by
detecting the presence or absence of a molecular marker for the
microorganism in the sample. In particular, the molecular markers
to be detected include proteins, such as enzymes that are specific
to a species of microorganism.
[0009] In one aspect, the invention features a method for detecting
the presence or absence of a microorganism, for example, E. coli in
a sample, comprising the steps of contacting the sample with a
detectably labeled substrate for an enzyme produced and/or secreted
by the microorganism, under conditions that result in modification
of the substrate by the enzyme; and detecting the modification or
the absence of the modification of the substrate. Modification of
the substrate indicates the presence of the microorganism in the
sample, and the absence of modification of the substrate indicates
the absence of the microorganism in the sample. In particular, the
substrate can consist of labeled peptide that is cleaved by a
protease enzyme to give a signal that can be detected. Furthermore,
this peptide can be designed with a particular sequence of amino
acid residues extending from one end of the original substrate
peptide as a "tag" for use in covalently coupling the substrate to
a surface.
[0010] In another aspect, the present invention features a method
for diagnosing the presence or absence of an infection in a
subject, comprising the steps of a) contacting a sample obtained
from a wound in a subject with a detectably labeled substrate for
an enzyme produced and/or secreted by a microorganism, for example,
E. coli, under conditions that result in modification of the
substrate by the enzyme; and b) detecting a modification or the
absence of a modification of the substrate. Modification of the
substrate indicates the presence of an infection in the subject,
and the absence of modification of the substrate indicates the
absence of an infection in the subject.
[0011] In yet another aspect, the present invention features a
method for diagnosing the presence or absence of a wound infection
in a subject, comprising the steps of a) contacting a subject with
a detectably labeled substrate for an enzyme produced and/or
secreted by a microorganism, for example, E. coli, under conditions
that result in modification of the substrate by the enzyme; and b)
detecting a modification or the absence of a modification of the
substrate. Modification of the substrate indicates the presence of
a wound infection in the subject, and the absence of modification
of the substrate indicates the absence of a wound infection in the
subject.
[0012] In another aspect, the invention features a biosensor for
detecting the presence or absence of a microorganism, for example.
E. coli, comprising a solid support and a detectably labeled
substrate for an enzyme produced and/or secreted by the
microorganism, wherein the substrate is attached to the solid
support.
[0013] In still another aspect, the present invention features a
kit for detecting an infection, comprising a biosensor for
detecting the presence or absence of a microorganism in a sample,
and one or more reagents for detecting the presence of the
microorganism that is the causative agent of the infection. For
example, the reagent can be used to detect an enzyme secreted by
the microorganism. In particular, the reagent can be used to detect
the modification of the substrate of the biosensor.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0014] FIG. 1 is a graph of the cleavage of target substrate ecot1
(T1) (relative fluorescence) in samples containing various
bacteria, as indicated. All bacterial samples are directly from
culture and include cells and media. (Legend abbreviations:
Buffer=20 mM tris buffer (pH 7.4) with 150 mM NaCl, Peptide
T1=labeled peptide substrate)
[0015] FIG. 2 is a graph of the cleavage of target substrate ecot2
(T2) (relative fluorescence) in samples containing various
bacteria, as indicated. All bacterial samples are directly from
culture and include cells and media. (Legend abbreviations:
Buffer=20 mM tris buffer (pH 7.4) with 150 mM NaCl, Peptide
T2=labeled peptide substrate)
[0016] FIG. 3 is a graph of the cleavage of target substrate ecot2
(T2) (relative fluorescence) in samples containing various
bacteria, as indicated, plus fetal bovine serum (FBS). All
bacterial samples are directly from culture and include cells and
media. (Legend abbreviations: Buffer=20 mM tris buffer (pH 7.4)
with 150 mM NaCl, Peptide T2=labeled peptide substrate, FBS=fetal
bovine serum)
[0017] FIG. 4 is a graph of the cleavage of target substrate ecot2
(T2) (relative fluorescence) in simulated wound fluid samples
containing various bacteria plus bovine serum albumin (BSA). All
bacterial samples are directly from culture and include cells and
media. (Legend abbreviations: Buffer=20 mM tris buffer (pH 7.4)
with 150 mM NaCl. Peptide T2=labeled peptide substrate)
[0018] FIG. 5 is a graph of cleavage of protease substrate T3
(relative fluorescence) over time in samples containing buffer,
buffer plus T3, buffer plus T3C (crude peptide), or culture
including cells and media from Pseudomonas, E. coli, S. aureus
(Staph aureus), S. epidermidis (Staph epidermidis), S. Salivarius
(Strep salivarius), S. pyogenes (Strep pyogenes), Enterococcus, or
Serratia.
DETAILED DESCRIPTION OF THE INVENTION
[0019] As part of their normal growth processes, many
microorganisms secrete a number of enzymes into their growth
environment. These enzymes have numerous functions including, but
not limited to, the release of nutrients, protection against host
defenses, cell envelope synthesis (in bacteria) and/or maintenance,
and others as yet undetermined. Many microorganisms also produce
enzymes on their cell surface that are exposed to (and interact
with) the extracellular environment. Many of these enzymes are
specific to the microorganism that secretes them, and as such, can
serve as specific markers for the presence of those microorganisms.
A system that can detect the presence of these enzymes that are
produced and/or secreted can equally serve to indicate the presence
of the producing/secreting microorganism. Alternatively, a system
that can detect the absence of these enzymes that are produced
and/or secreted can equally serve to indicate the absence of the
producing/secreting microorganism. Such a detection system is
useful for detecting or diagnosing an infection. As used herein, an
"infection" means a disorder caused by exposure to a pathogenic
microorganism. In one example, the microorganism is E. coli. In
another example, the disorder is a wound infection, an intestinal
disorder, food poisoning, a kidney disorder, or a urinary tract
infection.
[0020] A microorganism detection test system, as described herein
can be tailored to detect one specific microorganism, for example,
E. coli by identifying a protein such as a secreted enzyme specific
to the microorganism to be detected. Alternatively, a test system
can be designed to simultaneously identify more than one
microorganism species (for example, at least 2, at least 5, or at
least 10 different microorganism species), such as those that
commonly cause infections. Identifying those enzymes that are
common to certain classes of pathogenic microorganisms, but which
are not present in non-pathogenic microorganisms is one way to
achieve this goal. Such enzymes can be identified, for example,
with a computer based bioinformatics screen of the microbial
genomic databases. By using enzymes as the basis for detection
systems, sensitive tests can be designed, since even a very small
amount of enzyme can catalyze the turnover of a substantial amount
of substrate.
[0021] The present invention pertains to the identification of
bacterial proteins that are specific for microorganisms that are
the causative agent of an infection. The presence of a pathogenic
bacterium can be detected by designing a synthetic substrate that
will specifically react with an enzyme that is present on the
surface of the cell or secreted. These synthetic substrates can be
labeled with a detectable label such that under conditions wherein
their respective enzymes specifically react with them, they undergo
a modification, for example, a visible color change that is
observed.
[0022] The enzymes that are used in the bacteria detection method
of the present invention are preferably infection-specific enzymes.
As used herein, an infection specific enzyme is an enzyme produced
and/or secreted by a pathogenic bacteria, but is not produced
and/or secreted by a non-pathogenic bacteria. Examples of
pathogenic bacteria include, but are not limited to staphylococcus
(for example, Staphylococcus aureus, Staphylococcus epidermidis, or
Staphylococcus saprophyticus), streptococcus (for example,
Streptococcus pyogenes, Streptococcus pneumoniae, or Streptococcus
agalactiae), enterococcus (for example, Enterococcus faecalis, or
Enterococcus faecium), corynebacteria species (for example,
Corynebacterium diptheriae), bacillus (for example, Bacillus
anthracis), listeria (for example, Listeria monocytogenes),
Clostridium species (for example, Clostridium peifringens,
Clostridium tetanus, Clostridium botulinum, Clostridium difficile),
Neisseria species (for example, Nelsseria meningitidis, or
Neisseria gonorrhoeae), E. coli, Shigella species, Salmonella
species, Yersinia species (for example, Yersinia pestis, Yersinia
pseudotuberculosis, or Yersinia enterocolitica), Vibrio cholerae
Campylobacter species (for example, Campylobacter jejuni or
Campylobacter fetus), Helicobacter pylori, pseudomonas (for
example, Pseudomonas aeruginosa or Pseudomonas mallel), Haemophilus
influenzae, Bordetella pertussis, Mycoplasma pneumoniae, Ureaplasma
urealyticum, Legionella pneumophila, Treponema pallidum, Leptospira
interrogans, Borrelia burgdorferi, mycobacteria (for example,
Mycobacterium tuberculosis), Mycobacterium leprae, Actinomyces
species, Nocardia species, chlamydia (for example, Chlamydia
psittaci, Chlamydia trachomatis, or Chlamydia pneumoniae),
Rickettsia (for example, Rickettsia ricketsii, Rickettsia
prowazekii or Rickettsia alean), brucella (for example, Brucella
abortus, Brucella melitensis, or Brucella suis), Proteus mirabilis,
Serratia marcescens, Enterobacter docae, Acetinobacter anitratus,
Klebsiella pneumonia and Francisella tularensis. Preferably, the
infection-specific bacteria is staphylococcus, streptococcus,
enterococcus, bacillus, Clostridium species, E. coli, yersinia,
pseudomonas, Proteus mirabilis, Serratia marcescens, Enterobacter
docae, Acetinobacter anitratus, Klebsiella pneumoniae or
Mycobacterium leprae. For example, the infection-specific enzyme
can be produced and/or secreted by Staphylococcus aureus,
Staphylococcus epidermidis, Streptococcus pyogenes, Pseudomonas
aeruginosa, Enterococcus faecalis, Proteus mirabilis, Serratia
marcescens, Enterobacter docae, Acetinobacter anitratus, Klebsiella
pneumonia and/or Escherichia coli.
[0023] Preferably, the enzyme is one or more of the following;
phospholipase A protein, outer membrane protein T (ompT), or other
omp proteins. The sequences of these proteins can be obtained by
carrying out searches on protein sequence databases, for example,
GenBank, and one skilled in the art can carry out such a search.
Gene encoding such proteins can also be cloned using cloning
techniques known to one of skill in the art.
[0024] Substrates for use in the present invention include any
molecule. Either synthetic or naturally-occurring that can interact
with an enzyme of the present invention. Examples of substrates
include those substrates described herein, as well as substrates
for these enzymes that are known in the art. Other examples of
substrates include ecot1 (T1) derived fluorescent peptides, for
example, Edans-DSRPVRRRRRPRVSK-Dabcyl (SEQ ID NO: 1) or ecot2 (T2)
derived fluorescent peptides. for example, Edans-KVSRRRRRGGD-Dabcyl
(SEQ ID NO: 2), which can be cleaved by the ompT protein of
pathogenic E. coli. Such substrates described herein can be
obtained from commercial sources, e.g., Sigma (St. Louis, Mo.), or
can be produced, e.g., isolated or purified, or synthesized using
methods known to those of skill in the art.
[0025] The enzymes of the present invention can modify substrates,
for example, proteins or polypeptides by cleavage, and such
modification can be detected to determine the presence or absence
of a pathogen in a sample. One method for detecting modification of
a substrate by an enzyme is to label the substrate with two
different dyes, where one serves to quench the fluorescence of the
other dye by fluorescence resonance energy transfer (FRET) when the
molecules, for example, dyes or colorimetric substances are in
close proximity, and is measured by detecting changes in
fluorescence.
[0026] FRET is the process of a distance dependent excited state
interaction in which the emission of one fluorescent molecule is
coupled to the excitation of another. A typical acceptor and donor
pair for resonance energy transfer consists of 4-[[-dimethylamino)
phenyl]azo]benzoic acid (Dabcyl) and
5-[(2-aminoethylamino]naphthalene sulfonic acid (Edans). Edans is
excited by illumination with 336 nm light, and emits a photon with
wavelength 490 nm. If a Dabcyl moiety is located within angstroms
of the Edans, this photon will be efficiently absorbed. Dabcyl and
Edans will be attached to opposite ends of a peptide substrate. If
the substrate is intact, FRET will be very efficient. If the
peptide has been cleaved by an enzyme, the two dyes will no longer
be in close proximity and FRET will be inefficient. The cleavage
reaction can be followed by observing either a decrease in Dabcyl
fluorescence or an increase in Edans fluorescence (loss of
quenching).
[0027] If the substrate to be modified is a protein, peptide, or
polypeptide, the substrate can be produced using standard
recombinant protein techniques (see for example, Ausubel et al.,
"Current Protocols in Molecular Biology," John Wiley & Sons,
(1998), the entire teachings of which are incorporated by reference
herein). In addition, the enzymes of the present invention can also
be generated using recombinant techniques. Through an ample supply
of enzyme or its substrate, the exact site of modification can be
determined, and a more specific substrate of the enzyme can be
defined, if so desired. This substrate can also be used to assay
for the presence of the pathogenic bacteria.
[0028] The substrates are labeled with a detectable label that is
used to monitor interactions between the enzyme and the substrate
and detect any substrate modifications, for example, cleavage of
the substrate or label resulting from such interactions. Examples
of detectable labels include various dyes that can be incorporated
into substrates, for example, those described herein, spin labels,
antigen or epitope tags, haptens. enzyme labels, prosthetic groups,
fluorescent materials, chemiluminescent materials, bioluminescent
materials, and radioactive materials. Examples of suitable enzyme
labels include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, and acetylcholinesterase; examples of
suitable prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride and
phycoerythrin; an example of a chemiluminescent material includes
luminol; examples of bioluminescent materials include lueiferase,
lueiferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S, and .sup.3H. Other
examples of detectable labels include Bodipy, Pyrene, Texas Red,
Edans, Dansyl Aziridine, IATR and fluorescein. Succirnidyl esters,
isothiocyanates, and iodoacetamides of these labels are also
commercially available. When detectable labels are not employed,
enzymatic activity can be determined by other suitable methods,
example, detection of substrate cleavage through electrophoretic or
methods known to one skilled in the art.
[0029] One example of a preferred detectable label is a chromogenic
dye that allows monitoring of the hydrolysis of the substrate by
the microorganism. An example of such a dye is para-nitrophenol.
When conjugated to a substrate molecule, this dye will remain
colorless until the substrate is modified by the secreted enzyme,
at which point it turns yellow. The progress of the
enzyme-substrate interaction can be monitored by measuring
absorbance at 415 nm in a spectrophotometer. Other dyes that
produce detectable modification, e.g., a visible color change, are
known to those of skill in the art.
[0030] The sample in which the presence or absence of a bacteria,
such as E. coli is detected, or an infection is diagnosed, can be,
for example, a wound, a body fluid, such as blood, urine, sputum,
or wound fluid (for example, pus produced at a wound site). The
sample can also be any article that bacteria may be contained
on/in, for example, a wound dressing, a catheter, a urine
collection bag, a blood collection bag, a plasma collection bag, a
disk, a scope, a filter, a lens, foam, cloth, paper, a suture,
swab, test tube. a well of a microplate, contact lens solutions,
food packaging material, or a swab from an area of a room or
building, for example, an examination room or operating room of a
healthcare facility, a bathroom, a kitchen, or a process or
manufacturing facility.
[0031] The present invention also features a biosensor for
detecting a (one or more, for example, at least 2, at least 5, at
least 10, at least 20, at least 30, at least 50, at least 75, or at
least 100) marker protein enzyme(s) described herein and for
notifying a consumer of the presence of the marker protein. A
biosensor for use in healthcare settings or home use to detect
infections comprising a (one or more) specific substrate(s) that is
coupled to a solid support that is proximal to a wound or other
body fluid that is being monitored for bacterial contamination is
provided. Preferably, the substrate is covalently bound to a label
and thus has a detection signal that upon proteolysis of the
substrate-label bond indicates the presence of the bacteria. Such a
biosensor can also be used in food preparation settings to detect
for products that are contaminated with bacteria.
[0032] The biosensor is made by first determining the specific
substrate of a specific enzyme characteristic of the bacteria to be
detected. The determined specific substrate is labeled with one or
more, and preferably, a plurality of detectable labels, for
example, chromatogenic or fluorescent leaving groups. Most
preferably, the labeling group provides a latent signal that is
activated only when the signal is proteolytically detached from the
substrate. Chromatogenic leaving groups include, for example,
para-nitroanalide groups. Should the substrate come into contact
with an enzyme secreted into a wound or other body fluid by
bacteria or presented on the surface of a bacterial cell, the
enzyme modifies the substrates in a manner that results in
detection of such a modification, for example, a change in
absorbance, which can be detected visually as a change in color
(for example, on the solid support, such as a wound dressing), or
using spectrophotometric techniques standard in the art.
[0033] The biosensor of the present invention also can comprise one
or more substrates (for example, at least 2, at least 5, at least
10, at least 20, at least 30, at least 50, at least 75, or at least
100 substrates) for produced and/or secreted enzymes of pathogenic
bacteria. The biosensor is a solid support, for example, a wound
dressing (such as a bandage, or gauze), any material that needs to
be sterile or free of microbial contamination, for example, a
polymer, disk, scope, filter, lens, foam, cloth, paper, or sutures,
or an article that contains or collects the sample (such as a urine
collection bag, blood or plasma collection bag, test tube,
catheter, swab, or well of a microplate).
[0034] Typically, the solid support is made from materials suitable
for sterilization if the support directly contacts the wound or
infected area or sample. In one embodiment of the present
invention, the biosensor can be directly contacted with the wound
or infected area. In some instances, a sterile covering or layer is
used to prevent contamination of the wound or body fluid upon such
direct contact. If such sterile coverings are used, they will have
properties that make them suitable for sterilization, yet do not
interfere with the enzyme/substrate interaction. Preferably, the
portion of the biosensor that comes into contact with the wound is
also nonadherent to permit easy removal of the biosensor from the
sample surface. For example, if the biosensor comprises a wound
dressing, the dressing contacts the wound for a time sufficient for
the enzyme substrate to react and then the dressing is removed from
the wound without causing further damage to the wound or
surrounding tissue.
[0035] Substrates suitably labeled with detectable labels, for
example, a chromogenic dye, and attached or incorporated into a
sensor apparatus, can act as indicators of the presence or absence
of pathogenic bacteria that secrete the aforementioned enzymes.
When more than one substrate is utilized, each may be labeled so as
to distinguish it from another (for example, using different
detectable labels) and/or each may be localized in a particular
region on the solid support.
[0036] Substrates with hydrophobic leaving groups can be
non-covalently bound to hydrophobic surfaces. Alternatively
hydrophilic or hydrophobic substrates can be coupled to surfaces by
disulfide or primary amine, carboxyl or hydroxyl groups. Methods
for coupling substrates to a solid support are known in the art.
For example, fluorescent and chromogenic substrates can be coupled
to solid substrates using non-essential reactive termini such as
free amines, carboxylic acids or SH groups that do not affect the
reaction with the pathogens. Free amines can be coupled to carboxyl
groups on the substrate using, for example, a 10 fold molar excess
of either N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) or
N-cyclohexyl-N'-2-(4'-methyl-morpholinium)ethyl
carbodiimide-p-toluene sulphonate (CMC) for 2 hrs at 4.degree. C.
in distilled water adjusted to pH 4.5 to stimulate the condensation
reaction to form a peptide linkage.
[0037] SH groups can be reduced with DTT or TCEP and then coupled
to a free amino group on a surface with N-e-Maleimidocaproic acid
(EMCA, Griffith et al., FEBS Lett. 134:261-263, 1981). Example of
substrates are provided herein.
[0038] The polypeptides of the invention also encompass fragments
and sequence variants of the polypeptide substrates described
herein. Variants include a substantially homologous polypeptide
encoded by the same genetic locus in an organism, i.e., an allelic
variant, as well as other variants. Variants also encompass
polypeptides derived from other genetic loci in an organism, but
having substantial homology to a polypeptide substrate described
herein Variants also include polypeptides substantially homologous
or identical to these polypeptides but derived from another
organism, i.e., an ortholog. Variants also include polypeptides
that are substantially homologous or identical to these
polypeptides that are produced by chemical synthesis. Variants also
include polypeptides that are substantially homologous or identical
to these polypeptides that are produced by recombinant methods.
[0039] The percent identity of two amino acid sequences can be
determined by aligning the sequences for optimal comparison
purposes (e.g., gaps can be introduced in the sequence of a first
sequence). The amino acids at corresponding positions are then
compared, and the percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., identity=# of identical positions/total # of
positions.times.100). In certain embodiments, the length of the
amino acid sequence aligned for comparison purposes is at least
30%, preferably, at least 40%, more preferably, at least 60%, and
even more preferably, at least 70%, 80%, 90%, or 100% of the length
of the reference sequence. The actual comparison of the two
sequences can be accomplished by well-known methods, for example,
using a mathematical algorithm. A preferred, non-limiting example
of such a mathematical algorithm is described in Karlin et al.,
Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). Such an algorithm
is incorporated into the BLAST programs (version 2.2) as described
in Schaffer et al. (Nucleic Acids Res., 29:2994-3005, 2001). When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs can be used. In one embodiment, the
database searched is a non-redundant (NR) database, and parameters
for sequence comparison can be set at: no filters; Expect value of
10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an
Existence of 11 and an Extension of 1.
[0040] In another embodiment, the percent identity between two
amino acid sequences can be accomplished using the GAP program in
the GCG software package (Accelrys Inc., San Diego, Calif.) using
either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of
12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet
another embodiment, the percent identity between two nucleic acid
sequences can be accomplished using the GAP program in the GCG
software package (Accelrys Inc.), using a gap weight of 50 and a
length weight of 3.
[0041] Other preferred sequence comparison methods are described
herein.
[0042] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the
polypeptide, e.g., the ability to act as a substrate for an E. coli
specific protease. Similarity is determined by conserved amino acid
substitution. Such substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Conservative substitutions are likely to be
phenotypically silent. Typically seen as conservative substitutions
are the replacements, one for another, among the aliphatic amino
acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues
Ser and Thr; exchange of the acidic residues Asp and Glu;
substitution between the amide residues Asn and Gln; exchange of
the basic residues Lys and Arg; and replacements among the aromatic
residues Phe and Tyr. Guidance concerning which amino acid changes
are likely to be phenotypically silent are found in Bowie el al.,
Science 247: 1306-1310, 1990).
[0043] Functional variants can also contain substitution of similar
amino acids that result in no change or an insignificant change in
function. Alternatively, such substitutions may positively or
negatively affect function to some degree. Non-functional variants
typically contain one or more non-conservative amino acid
substitutions, deletions, insertions, inversions, or truncation or
a substitution, insertion, inversion, or deletion in a critical
residue or critical region, such critical regions include the
proteolytic cleavage site for an infection-specific protease.
[0044] Amino acids in a polypeptide of the present invention that
are essential for cleavage by an E. coli specific protease can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science, 244: 1081-1085, 1989). The latter procedure introduces a
single alanine mutation at each of the residues in the molecule
(one mutation per molecule).
[0045] The invention also includes polypeptide fragments of the
peptide substrates or functional variants thereof. The present
invention also encompasses fragments of the variants of the
polypeptides described herein. Useful fragments include those that
retain the ability to act as substrates for an infection-specific
protease.
[0046] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the polypeptide fragment and an
additional region fused to the carboxyl terminus of the
fragment.
[0047] The biosensors of the present invention can be used in any
situation where it is desirable to detect the presence or absence
of bacteria, and in particular, pathogenic bacteria. For example,
bacteria that collects on work surfaces in food manufacturing or
food preparation facilities, or health care facilities, and in
particular in operating rooms can be detected with a biosensor as
described herein. A substrate, or more than one substrate, that can
be modified by an enzyme secreted by or presented on the surface of
a bacteria is labeled and covalently bound to a collector
substrate, such as cotton fibers on the tip of a swab. When more
than one substrate is utilized, each may be labeled so as to
distinguish it from another (for example, using different
detectable labels) and/or each may be localized in a particular
region on the solid support. The swab tip is used to wipe the
surface suspected of being contaminated by bacteria. The swab tip
is placed in a medium and incubated using conditions that allow
modification of the labeled substrate if an enzyme specific for the
bound, labeled substrate(s) is present.
[0048] The present invention also features a kit for detecting
infection-specific bacteria as described herein. The kit can
comprise a solid support, for example, having a plurality of wells
(e.g., a microtiter plate). to which a detectably labeled substrate
is linked, coupled, or attached. A means for providing one or more
buffer solutions is provided. A negative control and/or a positive
control can also be provided. Suitable controls can easily be
derived by one of skill in the art. A sample suspected of being
contaminated by a pathogen described herein is prepared using the
buffer solution(s). An aliquot of the sample, negative control, and
positive control is placed its own well and allowed to react. Those
wells where modification of the substrate. for example, a color
change is observed are determined to contain a microbial pathogen.
Such a kit is particularly useful for detecting an infection in a
subject.
[0049] Also encompassed by the present invention is a kit that
comprises a biosensor, such as a packaged sterilized wound dressing
or a sensor for food packaging material, and any additional
reagents necessary to perform the detection assay.
[0050] A method for developing an assay for detecting a pathogenic
bacteria that produces at least one enzyme that is secreted by the
cell or present on the surface of the cell and a method for using
the assay to detect pathogenic bacteria producing the enzyme(s) now
follows: [0051] Step 1) Define an amino acid sequence that uniquely
identifies the prokaryotic microorganism of interest. Alternatively
a (one or more) amino acid sequence that is unique to a specific
group of pathogens, for example, infection-specific pathogens can
be determined.
[0052] Select an amino acid sequence, for example, a protein,
peptide, or polypeptide (marker sequence) that uniquely
characterizes or marks the presence of the microorganism or group
of microorganisms (for example, infection-specific pathogens) of
interest. The selection can be performed utilizing a bioinfomatic
approach, for example, as described in detail below. One or more
amino acid sequences that are unique to a specific prokaryotic
microorganism are determined. [0053] Step 2) Obtain sufficient
enzyme to determine conditions facilitating optimal modification of
a substrate by the enzyme.
[0054] Isolate the enzyme from the extracellular medium in which
the pathogenic bacteria to be assayed is growing, or from the cell
membrane of the bacteria, using standard protein purification
techniques, described, for example, in Ausubel (supra).
[0055] Alternatively. if the genetic sequence encoding the enzyme
or the location of the genetic sequence encoding the enzyme are
unknown, isolate and clone the genetic sequence encoding the marker
amino acid of Step 1, or, first determine the genetic sequence, and
then proceed as before. [0056] Step 3) Determine the conditions for
growth of the prokaryotic organism and for the production of an
enzyme presented on the surface of the cell or secreted by the
cell.
[0057] Determine medium required for growth of the specific
prokaryotic microorganism of interest and for expression of its
unique active enzyme into the medium. Also determine whether a
second molecule, for example, an enzyme is required to convert the
specific enzyme from an inactive precursor form to an active form.
To determine if the enzyme has been secreted in an active form, a
sample of the bacterial culture is provided with chosen potential
substrates and cleavage of these substrates is determined. This can
be done, for example, by combining the bacteria that produce the
enzyme with the substrate in the appropriate media and incubating
at 37.degree. C. with gentle shaking. At preset times (0.1, 0.3,
1.0, 3.0, 5.0, 24 and 48 hours) the samples are centrifuged to spin
down the bacteria, and a small aliquot is removed for an SDS-PAGE
gel sample. After completion of the time course, the samples are
run on a 10-15% gradient SDS-PAGE minigel. Then, the proteins are
transferred to Immobilon Pseq (Transfer buffer, 10% CAPS, 10%
methanol pH 11.0, 15 V for 30 minutes) using a Bio-Rad semi-dry
transblotting apparatus. Following transfer of the proteins, the
blot is stained with Coomassie blue R-250 (0.25% Coomassie
Brilliant Blue R-250, 50% methanol, 10% acetic acid) and destained
(high destain for 5 minutes, 50% methanol, 10% acetic acid; low
destain until complete, 10% methanol, 10% acetic acid) followed by
sequencing from the N-terminal. Alternatively, the samples can be
run on a mass spectrometer in order to map the sites of proteolytic
cleavage using a Voyager Elite Mass spectrometer (perceptive
Biosystems, Albertville, Minn.). [0058] Step 4) Identify any
specific substrate(s) of the active enzyme protease. Examples of
potential substrates include proteins, peptides, polypeptides,
lipids, and peptidoglycan subunits. Label each substrate with a
detectable label, for example, a detectable label described herein,
or any other detectable label known in the art. [0059] Step 5)
Increase the specificity of the enzyme-substrate interaction
(optional) by determining the active or binding site of the enzyme
(for example, using FRET as described above), then determining the
genetic sequence useful for producing the active or binding site,
and cloning the determined genetic sequence to generate a more
specific substrate. [0060] Step 6) Provide a biosensor comprising
one or more of the detectably labeled substrates identified above
for detection of the protease of the pathogenic bacteria of
interest.
[0061] The substrate can be attached to solid support, for example,
a wound dressing, or an article that holds the enzyme and
substrate, for example, a body fluid collection tube or bag, a
microplate well, or a test tube. The solid support, if desired, can
provide a plurality of derivatized binding sites for coupling to
the substrate, for example, succimidyl ester labeled primary amine
sites on derivatized plates (Xenobind plates, Xenopore, Hawthorne,
New jersey).
[0062] Optionally, unoccupied reactive sites on the solid support
are blocked by coupling bovine serum albumin, or the active domain
of p26 thereto. p26 is an alpha-crystallin type protein that is
used in this case to reduce non-specific protein aggregation. The
ability of the p26 protein to refold heat denatured citrate
synthetase before and after coupling to the surface of the food
packaging is used as a control for determining p26 activity.
Alpha-crystallin type proteins were recombinantly produced using
standard recombinant DNA technologies (see Ausubel, supra).
Briefly, the plasmid containing the beta sheet-charged core domain
of p26 is electroporated into electrocompetent BL21 (DE3) cells
(Bio-Rad E. coli pulser). The cells are grown up to an OD.sub.600
of 0.8, then induced with 1 mM IPTG for 4 hours. The cells are spun
down, and sonicated in low buffer (10 mM Tris, pH 8.0, 500 mM NaCl,
50 mM Imidizole) to lyse (Virsonic, Virtis, Gardiner, N.Y.). The
lysate is spun down at 13,000.times.g for 10 minutes, and the
supernatant 0.45 and 0.2 .mu.m filtered. This filtrate is loaded
onto a Ni-NTA superose column (Qiagen, Valencia, Calif., cat
#30410). High buffer (10 mM Tris pH 8.0, 500 mM NaCl, 250 mM
Imidizole) is used to elute the protein.
[0063] Allow the enzyme(s) to come into contact with the
substrate(s), and monitor the reaction for a modification in the
detectably labeled substrate, as described herein. Modification of
the substrate indicates that the enzyme produced/secreted by the
bacteria is present in the reaction. In addition, the absence of
modification of the substrate indicates that the enzyme is not
present in the sample. If the bacteria or enzyme is from a wound or
other infected area. modification of the substrate indicates that
the bacteria is present in the wound or infected area. and that the
wound or area is infected, while the absence of modification of the
substrate indicates that the particular bacteria is not present in
the wound or area, and that the wound or area is not infected with
that particular bacteria.
EXAMPLES
[0064] The present invention will now be illustrated by the
following Examples, which are not intended to be limiting in any
way.
Example 1
Detection of the Presence of E. coli in a Sample E. coli Assay
Development
[0065] The Gram-negative bacterium Escherichia coli is the best
characterized human pathogen and is known to secrete very few
molecules unless specifically required for virulence. The virulent
strains include those likely to cause food poisoning (0157:H7),
intestinal disorders (EHEes) or urinary tract infections (UTIs).
However. most strains of E. coli can harmlessly coexist with humans
and are not likely to cause disease under normal circumstances.
[0066] Although many of the genes are common to other bacteria. E.
coli has developed some unique means of coexistence. A search of
the E coli K-12 genome by subtraction of several other pathogenic
and non-pathogenic bacteria provides a list of genes that are
unique to this organism. The listing obtained includes the outer
membrane proteins phospholipase A, outer membrane protein T (ompT)
and several other omp genes.
[0067] The gene ompT encodes an enzyme that is found on the outer
surface of the cell membrane and is used to protect the cell from
strongly cationic antimicrobial peptides (defensins) produced by
humans. The protein OmpT is a membrane bound protease that has been
shown to efficiently cleave protamines (salmon milt). The enzyme
binds positively charged proteins and peptides and cleavage occurs
preferentially at a site between two positively charged
residues.
[0068] The peptide substrates used here were labeled with the
fluorescent probe edans
(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) and the
quencher dye molecule dabcyl
((4-(4-(dimethylamino)phenyl)azo)benzoic acid). The labeled
peptides ecot1 (T1) and ecot2 (T2) sequences used are as
follows:
TABLE-US-00001 (SEQ ID NO: 1) (T1) Edans - DSRPVRRRRRPRVSK - Dabcyl
(SEQ ID NO: 2) (T2) Dabcyl - KVSRRRRRGGD - Edans
[0069] The bacteria were grown in an incubator overnight at
37.degree. C. in 5 mL BHI (Brain Heart Infusion) media. Each of the
resulting cultures was split into two samples. One was used as a
culture, and the other was spun down by centrifugation and the
supernatant was collected. The assays were run in 20 mM tris buffer
(pH 7.4) with 150 mM NaCl added. The reaction was carried out with
5 .mu.L of culture or supernatant and 5 .mu.L of peptide substrate
(10 mg/mL in water) in 100 .mu.L total volume at 37.degree. C. The
reaction was followed on a fluorimetric plate reader using an
excitation wavelength of 355 nm and an emission wavelength of 485
nm.
[0070] The first set of experiments was performed by addition of
the bacterial culture directly into the assay solution. The
protease OmpT is a membrane bound protein and would not be expected
to be found secreted into the media. The first assay to be run used
the peptide ecot1 (T1) as substrate. The results are shown in FIG.
1.
[0071] As shown in FIG. 1, protease activity was observed for both
E. coli and Pseudomonas with the T1 peptide substrate. The same
protease assay was repeated under identical conditions for
substrate ecot2 (T2). The results are shown in FIG. 2.
[0072] As shown in FIG. 2, the sample containing E. coli cells
reacted with this substrate. This peptide appears to be both
efficient and selective for E. coli.
[0073] To test whether the protease is membrane associated, as
expected for E. coli OmpT, the protease assays were repeated with
the supernatants obtained from each bacterial culture. When peptide
substrate T1 was used with the bacterial culture supernatants, the
protease activity observed for Pseudomonas was still present, but
the activity associated with the E. coli cells was not present in
the supernatant. This indicates that the protease from Pseudomonas
is secreted into solution, but the E. coli protease observed here
is membrane bound and may be due to OmpT. When peptide substrate T2
was used with the bacterial culture supernatants, the peptide
substrate T2 did not show any reactivity with a secreted protease
from E. coli or any of the other bacteria tested. This indicates
that peptide T2 appears to be selective for the E. coli outer
membrane protease OmpT.
[0074] The T2 peptide substrate was further tested for cross
reactivity with the types of conditions and molecules that may be
present in a wound environment. A fluid that may be present in a
wound, at least initially, is serum. In order to test for
reactivity with serum the reaction buffer was modified to by
addition 5% fetal bovine serum and the protease assay was repeated,
using the T2 peptide substrate. The results are shown in FIG. 3. As
shown in FIG. 3, detection of the presence of E. coli in the E.
coli sample occurred in the presence of FBS.
[0075] The protease assay was also tested in a simulated wound
fluid buffer. The buffer was tris-buffered saline, as described
above, to which 5% (by weight) bovine serum albumin was added. The
protease assay was repeated, again using the T2 peptide substrate.
The results of this assay are shown in FIG. 4. As shown in FIG. 4,
the protease reactivity of the E. coli sample was not affected by
the simulated wound fluid buffer. Under these conditions the
peptide T2 appears to be a rapid and selective probe for the
detection of E. coli cells.
Example 2
Development of Biosensor Surfaces
[0076] The attachment of molecules to surfaces can be performed by
the use of several different types of interactions. Typically,
proteins can be attached to surfaces using hydrophobic,
electrostatic, or covalent interactions. There are many
commercially available membranes and resins with a variety of
surface properties. Surfaces can also be chemically modified to
provide the required surface properties.
[0077] Commercially available transfer membranes exist for protein
and peptide binding. They consist of positively and negatively
charged polymers such as ion exchange membrane disc filters and
resins. Nitrocellulose membranes offer hydrophobic and
electrostatic interactions. Glass fiber membranes offer a
hydrophobic surface that can easily be chemically modified to add
functional groups. There are also modified polymer membranes that
offer reactive functional groups that covalently bind proteins and
peptides.
[0078] It is also possible to utilize various functional groups on
membranes or resins and a crosslinking agent to covalently link to
proteins. Crosslinking reagents contain two reactive groups thereby
providing a means of covalently linking two target functional
groups. The most common functional groups to target on proteins are
amine, thiol, carboxylic acid, and alcohol groups that are used to
form intramolecular crosslinks. Crosslinking agents can be homo
bifunctional or heterobifunctional and a selection of crosslinking
agents of various lengths are commercially available.
[0079] Initially the peptides studied were designed as substrates
for bacterial assay development using fluorescence energy transfer
(Edans and Dabcyl) for detection. T2, which is selective for E.
coli. is an example of such a substrate, and is described
herein.
[0080] In order to develop substrates specifically for surface
immobilization, several versions of the T2 peptide were made. The
peptides were designed to include lysine groups (amine functional
group) at one end of the peptide in the case of T2. The addition of
two lysine groups (KK) at one end of the peptide serve as a "tag"
and provide ideal groups for attachment to surfaces through
techniques such as electrostatic interactions or through covalent
attachment. The peptide T4 was designed to include a cysteine group
(C) and three histidine groups (HHH) at one end. The addition of a
cysteine provides another ideal group or tag to perform covalent
attachments through the thiol group. The inclusion of three
histidine groups also provides the potential for attachment to
nickel resins.
[0081] The peptide sequence for T2 was modified as shown:
TABLE-US-00002 (SEQ ID NO: 2) T2 (dabcyJ-K)VSRRRRRGG(D-edans) (SEQ
ID NOS: 3 and 4) T3 KKAS(E-edans)VSRRRRRGO(K-dabcyl) (SEQ ID NOS: 5
and 6) T4 CHHHAS(E-edans)VSRRRRROO(K-dabcyl)
The pre-peptide tags were added to the original sequences to allow
for attachment to a surface.
[0082] The protease assay, described herein for detection of E.
coli was run with the modified version of T2, T3. Bacteria
(Pseudomonas, E. coli, S. aureus, S. epidermidis, S. salivarius, S.
pyogenes, Enterococcus, and Serratia) were grown in an incubator
overnight at 37.degree. C. in 5 mL BHI (Brain Heart Infusion)
media. The assays were run in 20 mM tris buffer (pH 7.4) with 150
mM NaCl added. The reaction was carried out with 7 .mu.L of culture
including cells and media and 3 .mu.L peptide substrate (5 mg/mL in
water) in 100 J.I.L total volume at 37.degree. C. The reaction was
followed on a fluorimetric plate reader using an excitation
wavelength of 355 nm and an emission wavelength of 485 nm. The
results are shown in FIG. 5. As shown in FIG. 5, this assay appears
to be specific for E. coli.
[0083] Metal chelate (affinity binding) interactions can provide a
stronger bond to biological molecules. A his-tag built into the
peptide substrate, for example T4 can be used to allow linkage to a
nickel binding resin. The resin is incubated with a suitable
culture, for example, E. coli for 30 minutes at 37.degree. C. After
centrifugation the buffer is removed and the pelleted resin is
imaged. The fluorescence produced by the peptide is then detected.
In an example of such a detection assay, E. coli was detected using
a biosensor in which a his-tagged T4 peptide was linked to a nickel
binding resin and subsequently exposed to E. coli cultures or
exposed to BHI media without bacteria.
[0084] Lysine peptide tags, for example, T3 can be used to link to
a surface such as UltraBind.TM. (pall Gelman Laboratory, Ann Arbor,
Mich.). UltraBind is a polyethersulfone membrane that is modified
with aldehyde groups for covalent binding of proteins. Proteins are
directly reacted with the UltraBind surface. It is also possible to
link proteins or peptides to the surface using cross linker chains.
For example, the carbodiimide, EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride) is
commonly used to link carboxylic acid groups to amines. The linking
of the peptide with a cross linking agent allows the choice of a
linker chain to extend the peptide off the surface of the membrane
while still covalently binding 10 it. The linking of the peptide
through a cross linker can be optimized to make the peptide
available to the bacterial enzymes. This allows for optimization of
the reaction time of the sensor since peptide availability is
directly related to this parameter.
[0085] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
6115PRTArtificial SequenceSynthetic peptide 1Asp Ser Arg Pro Val
Arg Arg Arg Arg Arg Pro Arg Val Ser Lys 1 5 10 15 211PRTArtificial
SequenceSynthetic peptide 2Lys Val Ser Arg Arg Arg Arg Arg Gly Gly
Asp 1 5 10 35PRTArtificial SequenceSynthetic peptide 3Lys Lys Ala
Ser Glu 1 5 410PRTArtificial SequenceSynthetic peptide 4Val Ser Arg
Arg Arg Arg Arg Gly Gly Lys 1 5 10 57PRTArtificial
SequenceSynthetic peptide 5Cys His His His Ala Ser Glu 1 5
610PRTArtificial SequenceSynthetic peptide 6Val Ser Arg Arg Arg Arg
Arg Gly Gly Lys 1 5 10
* * * * *